Name: quantification of the immunosuppressant tacrolimus on dried blood spots using lc-ms/ms
Uploaded: 2015-11-08T14:00:00.000+00:00
Duration: 8 min 38 s
Description: Watch this Scientific Journal Video about Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS at JoVE.com

This work was supported by the United States Federal Drug Administration (FDA) contract HHSF223201310224C and the United States National Institutes of Health/FDA grant 1U01FD004573-01.

De-identified blood samples from healthy individuals were from the University of Colorado Hospital (Aurora, Colorado). The use of de-identified blood bank samples for validation studies as well as for the preparation of calibrators and quality control samples was considered &#8220;exempt&#8221; by the Colorado Multi-institutional Review Board (COMIRB, Aurora, Colorado).
1. Preparation of References and Solutions
<ol>
	<li>Purchase tacrolimus and the internal standard D2,13C-tacrolimus from the vendors listed in the Materials List.
	<ol>
		<li>Prepare stock solutions in pure methanol at a concentration of 1 mg/ml for tacrolimus and a concentration of 10 &#181;g/ml for D2,13C-tacrolimus. Make stock solutions of reference materials based on three independent weightings. Aliquot stock solutions and store at -70 &#176;C or below.</li>
	</ol>
	</li>
	<li>Prepare solution to precipitate proteins and extract tacrolimus using methanol / 0.2 M ZnSO4 in water (7:3, v:v). This solution also contains the internal standard D2,13C-tacrolimus at a concentration of 2.5 ng/ml and is used for the extraction of all samples except for the extraction of blank samples (please see 1.3.3).
	<ol>
		<li>Prepare this protein precipitation solution freshly on each extraction day and set the expiration of the solution at 12 hr.</li>
	</ol>
	</li>
	<li>Preparation of calibration curve and quality control (QC) samples
	<ol>
		<li>Prepare stock solutions of tacrolimus by performing appropriate dilutions of the stock solution using pure methanol.</li>
		<li>To prepare calibrators and quality control samples, spike 20 &#181;l of appropriately diluted stock solution into EDTA whole blood, incubate at 37 &#176;C under gentle shaking in a water bath to allow for homogeneous distribution of tacrolimus into the cellular blood components for 20 min and aliquot into 1.5 ml polypropylene tubes with conical bottom and snap-on lids. Ensure that the relative volume of organic solvent does not exceed 5%.</li>
		<li>Spot 50 &#181;l of the spiked whole blood into the middle of each circle on the filter cards using a pipette.</li>
		<li>Dry the blood spots on filter cards at RT for 3 hr.</li>
		<li>Prepare tacrolimus calibration standards in human EDTA whole blood at tacrolimus concentrations of 1, 2.5, 5, 10, 25, and 50 ng/ml. Prepare a blank sample for extraction like the calibration standards with the protein precipitation solution containing the internal standard D2,13C-tacrolimus (&#8220;zero sample&#8221;).</li>
		<li>Prepare QC samples in human EDTA whole blood at concentrations of 0, 2, 4, 20, 40 ng/ml. Prepare a blank sample. In contrast to the QC samples that are extracted with precipitation containing the internal standard D2,13C-tacrolimus, extract this blank sample with protein precipitation solution that does not contain the internal standard D2,13C-tacrolimus (&#8220;blank sample&#8221;).</li>
	</ol>
	</li>
	<li>Collection of clinical samples
	<ol>
		<li>Collect dried blood spots as described in43,44.</li>
	</ol>
	</li>
</ol>
2. Extraction of Tacrolimus Dried Blood Spot Samples
<ol>
	<li>Visually inspect the dried blood spot to ensure acceptable sample quality and volume45.</li>
	<li>Punch center of the blood spot on the filter card with a 6-mm hole punch. 
	Note: The quality of punches may be monitored by weighing. A punched saturated filter disc weighs in average 5.02 mg &#177; 0.09 mg (range: 4.83- 5.14 mg, n=12).</li>
	<li>Place discs into 1.5 ml polypropylene tubes with conical bottom and snap-on lids.</li>
	<li>Add 20-30 bullets to each tube.</li>
	<li>Add 500 &#181;l of the protein precipitation solution (methanol: 0.2 M ZnSO4, 7:3, v:v with 2.5 ng/ml internal standard) into each tube. For the extraction of blank samples, use protein precipitation solution without the internal standard.</li>
	<li>Homogenize the discs in the bullet blender for 1 min (maximum speed, setting &#8220;10&#8221;).</li>
	<li>Shake samples at RT on multi-tube vortex (maximum speed, setting &#8220;10&#8221;) for 10 min.</li>
	<li>Centrifuge samples at 16,000 x g and 4 &#176;C for 10 min.</li>
	<li>Transfer the supernatants into glass HPLC vials equipped with a 300 &#181;l insert. Use pre-slit Teflon seals. 
	Note: Extracted samples may be stored at -20 &#176;C or below until LC-MS/MS analysis.</li>
</ol>
3. LC-MS/MS Analysis
<ol>
	<li>Load 100 &#181;l of the supernatant of the extracted sample onto the C8 cartridge extraction column and wash with a 7:3 ratio of 0.1% formic acid in water: acetonitrile at a flow of 5 ml/min for 1 min. The connections of the switching valve are shown in Figure 2 and the gradient run by the extraction pump in Table 1.</li>
	<li>Hereafter, activate the switching valve resulting in back-flush of the analytes from the pre-column onto the analytical column.</li>
	<li>Set the column thermostat to 65 &#176;C.</li>
	<li>Elute the analytes from the analytical column using the flow rates and gradient shown in Table 1.</li>
	<li>Connect the analytical column to a tandem mass spectrometer via the turbo electrospray ionization source. Adjust the key parameters of the mass spectrometer according to Table 2.</li>
	<li>Detect positive ions ([M+Na]+) in the multiple reaction mode (MRM). Use the following ion transitions for quantification: tacrolimus: m/z (mass/charge) = 826.6 &#8594; 616.2 and D2,13C-tacrolimus: m/z= 829.6 &#8594; 619.2. 
	Note: The total run time is 4.6 min.</li>
</ol>
4. Quantification
<ol>
	<li>For each run, generate a calibration curve based on the calibrators prepared in 1.3.5 and include in each analytical run.
	<ol>
		<li>Generate a calibration curve by plotting nominal concentrations versus response factor of analyte (Peak Area [Analyte] / Peak Area [internal standard]) using the mass spectrometer software.</li>
		<li>Fit the calibrators using a quadratic fit in combination with 1/X weighting.</li>
	</ol>
	</li>
	<li>To quantity tacrolimus in the dried blood spots integrate the tacrolimus and internal standard peaks in the extracted MRM chromatograms. Calculate the response factor for tacrolimus (Peak Area [Analyte] / Peak Area [internal standard]) and compare with the calibration curve using the mass spectrometry software.</li>
</ol>
5. Validation Procedures
<ol>
	<li>Lower limit of detection (LLOD) and lower limit of quantification (LLOQ).
	<ol>
		<li>Consider the lowest tacrolimus concentration with a peak-to-noise ratio of 4:1 as the lower limit of detection (LLOD). Define the lower limit of quantification (LLOQ) as the lowest concentration of the calibration curve with an accuracy equal to or better than &#177; 20% deviation from the nominal concentration and precision equal to or better than 20% (coefficient of variance).</li>
	</ol>
	</li>
	<li>Intra- and inter-day accuracies and precisions.
	<ol>
		<li>Test the accuracy and precision at four concentration levels of 2 ng/ml (QC1), 4 ng/ml (QC2), 20 ng/ml (QC3) and 40 ng/ml (QC4).</li>
		<li>Prepare the QC samples on each validation day in human EDTA whole blood, dry on filter cards, extract, and analyze as described above.</li>
		<li>Determine intra-day accuracy and precision with 6 samples per QC concentration level.</li>
		<li>Assess inter-day accuracy and precision over 20 days. Measure each QC level with 4 samples each day.</li>
		<li>Analyze two calibration curves together with the QC samples on each day.</li>
		<li>Calculate intra-day accuracy as the % of the nominal concentration (six samples per concentration level, please see 5.2.2). Calculate precision as the coefficient of variance (CV%).</li>
		<li>Consider intra-day accuracy acceptable if it falls into the acceptance limits 85% to 115% of the nominal concentration. Consider intra-day precision acceptable if it is equal to or better than a CV (coefficient of variance) of 15%.</li>
		<li>Calculate inter-day accuracy and precision as the mean for each QC concentration level analyzed over the 20 validation days.</li>
		<li>Consider mean inter-day accuracy acceptable if it falls into the acceptance limits 85% to 115% of the nominal concentration. Consider inter-day precision acceptable if it is equal to or better than a CV (coefficient of variance) of 15%.</li>
	</ol>
	</li>
	<li>Exclusion of matrix interferences.
	<ol>
		<li>For the exclusion of interferences that may be caused by matrix signals, analyze blank dried blood spots (8 different individuals, preferably 4 males and 4 females).</li>
		<li>Visually inspect ion chromatograms. If peaks within the retention time window of tacrolimus are detected, integrate and compare their areas under the curve with those of tacrolimus peaks in blank samples spiked with tacrolimus at the LLOQ. The area of peaks in the blank samples are not supposed to exceed 15% of those of tacrolimus at the LLOQ.</li>
	</ol>
	</li>
	<li>Ion suppression/ion enhancement.
	<ol>
		<li>Use a post-column infusion protocol as described45 to assess potential interference of ion suppression/ion enhancement caused by co-eluting matrix components.</li>
		<li>Infuse tacrolimus at a concentration of 10 &#181;g/ml dissolved in 0.1% formic acid: methanol (30:70, v/v) post-column at a rate of 10 &#181;l/min.
		<ol>
			<li>Connect a syringe pump via T-piece between the analytical column and the electrospray source of the mass spectrometer.</li>
			<li>Monitor the MS/MS signal intensity of the MRM transitions for tacrolimus and its internal standard (m/z = 826.6 &#8594; 616.2 and m/z = 829.6 &#8594; 619.2) after injection of extracted blank samples (n = 8 samples from different individuals). 
			Note: In the absence of ion suppression/ion enhancement the continuous signal caused by infusion of the analytes should not be affected by injection of the blank matrix, while ion suppression causes a dip of the signal and ion enhancement a peak.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Carry-over.
	<ol>
		<li>Assess potential carry-over by analyzing extracted blank samples after the highest calibrators (50 ng/ml, n = 6).</li>
		<li>Visually inspect ion chromatograms. If peaks within the retention time window of tacrolimus are detected, integrate and compare their areas under the curve with those of tacrolimus peaks in blank samples spiked with tacrolimus at the LLOQ. The area of peaks in the blank samples are not supposed to exceed 15% of those of tacrolimus at the LLOQ.</li>
	</ol>
	</li>
	<li>Extraction recoveries.
	<ol>
		<li>Determine recoveries by comparing the signals of the analytes after extraction of QC samples at all four concentration levels (n = 6 per concentration) with those of blank dried blood spots spiked with the corresponding amounts of tacrolimus after the extraction.</li>
		<li>Prepare four sets of QCs (concentration levels: 2, 4, 20, 40 ng/ml).</li>
		<li>Prepare another 4 sets of corresponding &#8220;recovery test samples&#8221; by spotting 50 &#181;l of blank EDTA whole blood onto the filter paper cards and drying for 2 hr.</li>
		<li>Hereafter, for both the QC and blank &#8220;recovery test samples&#8221;, cut out the entire blood spot on the filter card with scissors and place the resulting discs into a polypropylene tube with conical bottom and snap-on lid.</li>
		<li>Extract all samples.</li>
		<li>Transfer the supernatants (400 &#181;l) into glass HPLC vials.</li>
		<li>Add tacrolimus stock solution to the blank &#8220;extracted recovery test samples&#8221; to reach concentrations of 2, 4, 20 and 40 ng/ml (4 &#181;l of 200, 400, 2,000, 4,000 ng/ml tacrolimus stock solutions to 400 &#181;l of supernatant).</li>
		<li>After LC-MS/MS analysis, compare the signals in both QC samples and &#8220;recovery test samples&#8221; of the corresponding concentration (recovery% = signal samples spiked before extraction / signal samples spiked after extraction x 100).</li>
	</ol>
	</li>
	<li>Dilution integrity.
	<ol>
		<li>Establish dilution integrity using samples spiked with the analytes at 500, 250 and 100 ng/ml.</li>
		<li>After extraction, dilute samples using protein precipitation solution (1:10, n = 3 per concentration level).</li>
		<li>Calculate deviations from the nominal concentrations. Consider results that fall within 85%-115% of nominal acceptable.</li>
	</ol>
	</li>
	<li>Stabilities.
	<ol>
		<li>Investigate stabilities using the QC samples at all four concentration levels (n = 4 per concentration) analyzed at different time-points and under the different storage conditions.</li>
		<li>Compare results after storage with the nominal values. Consider results that fall within 85%-115% of nominal acceptable.</li>
		<li>Establish sample stability for 1 week at ambient temperature, 1 week at 4 &#176;C, 1 month at -20 &#176;C and 1 month at -80 &#176;C.</li>
		<li>Test freeze-thaw stability over three cycles (-20 &#176;C). Test extracted sample and autosampler stability by placing samples into the thermostatted autosampler adjusted to 4 &#176;C. Inject samples after 72 hr.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Although, as aforementioned, the concept of therapeutic drug and adherence monitoring of tacrolimus based on dried blood spots is attractive, there are analytical challenges that go beyond those typically associated with the LC-MS/MS analysis of tacrolimus in venous EDTA whole blood samples. These include, but are not limited to, the fact that the matrix is capillary whole blood soaked into the cotton linters material of the filter card material used here and the low blood volume (20 &#181;l). Nevertheless, high-throughput analysis in a central laboratory requires a fast and reliable extraction method that results in samples that lack matrix interferences and matrix effects in combination with a robust, specific and highly sensitive LC-MS/MS assay. Reliability of the assay is critical as there is usually not enough material on the already punched filter card left for re-analysis in case the extraction / LC-MS/MS analysis fails.
The filter cards used in the present study (for manufacturer details, please see Materials List) were chosen as these are an FDA approved class II device, are in compliance with NCCLS guidance LA4-A544 and are CE-marked in Europe. If completely filled, one circle on the Whatman 903 filter card holds &#8776;50 &#181;l blood46. However, the size of the blood drops collected by individual patients vary and training in the proper sampling technique is essential46.
The first important step of extracting a punched-out dried blood spot sample is homogenization. Based on our experience, the use of a bullet blender is more efficient and better reproducible than other methods used to enhance extraction efficiency such as sonication. The use of the bullet blender was essential to consistently achieve extraction recoveries above 90%. For reliability of the extraction procedure, it was also important to ensure that all centrifuges were temperature controlled (4 &#176;C) and that the vortexing step was not shorter than 10 min, which resulted in more variable and lower extraction recoveries. In addition, it is important that the methanol/ZnSO4 ratio is not altered as tacrolimus recovery is very sensitive to the correct composition of the protein precipitation solution.
The next challenge is to obtain a clean extract ideally devoid of materials that may cause matrix interferences and effects. Thus, a simple one-step protein precipitation step as often used for the extraction of tacrolimus from EDTA blood samples was not considered a viable option. After protein precipitation using ZnSO4 (including addition of an isotope-labeled internal standard), vortexing and centrifugation, the supernatants were injected into a 2D HPLC system and onto an online extraction column. Online column extraction using high flows of 5 ml/min on conventional pre-column cartridges using a simple 6-port switching valve for the analysis of tacrolimus have been described before47. The mobile phase was chosen so that tacrolimus and its internal standard concentrated in the front of the extraction column and did not migrate over the column during the cleanup step. The online extraction used in the present protocol had several advantages including injection of relatively large sample volumes without negatively affecting HPLC analysis. The back-flush after enriching the analytes on top of the extraction column (&#8220;peak focusing&#8221;) resulted in sharper peaks allowing for more reliable integration by the software algorithm especially for samples with low tacrolimus concentrations.48 The online cleanup step did not only remove potentially interfering matrix compounds, but also de-salted the sample. One important yet rarely discussed problem for high-volume, high throughput LC-MS/MS assays is the gradual loss of sensitivity of the LC-MS/MS system due to increasing contamination of the electrospray source during analysis of large batches. No significant matrix effects (ion suppression/ion enhancement) were observed. The negative effect of potential matrix effects were reduced/avoided by combination of the following: effective protein precipitation using methanol/ZnSO4, centrifugation after protein precipitation at 16,000 x g, high-flow online extraction, clear chromatographic separation of tacrolimus from potential interferences early eluting from the analytical column and the use of isotope-labeled tacrolimus as internal standard instead of structurally related internal standards such as ascomycin.
The lower limit of quantification was 1 ng/ml, and thus lower than that of most immunoassays that are currently frequently used for therapeutic drug monitoring of tacrolimus in EDTA blood samples. This lower limit of quantification is sufficient even for so-called low calcineurin inhibitor long-term immunosuppressive maintenance protocols.
In comparison with previously described LC-MS/MS assays to quantify tacrolimus in dried bloods spots29-34,36,39,41,42, the present assay matches or exceeds their performance in terms of lower limit of quantification, extraction recovery, accuracy and precision while avoiding potentially risky concepts such as one-step protein precipitation procedures and ultra-short chromatography times, which usually give acceptable results during the validation based on blood samples from healthy individuals. However, transplant patients are a highly complex group of patients who have diseases that affect the composition of blood and who take multiple medications. This makes it virtually impossible to exclude all potential interferences that may be present in individual patients during the validation and the only viable strategy is to set up the assay in a way that it minimizes the risk of such potential interferences. Dried blood spots have challenges such as the effect of the hematocrit on blood viscosity and thus the diffusion properties of the blood applied on filter paper49. This was not tested here again as such effects have already been described not to affect tacrolimus analysis in dried blood spots at hematocrits and tacrolimus concentrations within clinically reasonable limits29,32. Also stability of tacrolimus in dried blood spots at elevated temperatures has already been studied by others and tacrolimus in dried blood spots was found to be stable for 5 days at 37 &#176;C and even 60 &#176;C32, which is important for shipment of dried blood spots under not temperature-controlled conditions especially during summer.
This assay based on a combination of bullet blender homogenization, high-flow online column cleanup and LC-MS/MS analysis may provide a platform strategy for the development of bioanalytical assays for the quantification of other immunosuppressants, alone or simultaneous, as well as of other drugs in dried blood spot samples.

Tacrolimus is a potent immonosuppressant1-7 that has a macrolide structure8 (Figure 1). Due to cis-trans isomerism of the C-N bonds it forms two rotamers in solution9 that can be separated by reversed phase high-performance liquid chromatography (HPLC) Tacrolimus is lipophilic and soluble in alcohols (methanol: 653 g/L, ethanol: 355 g/L), halogenated hydrocarbons (chloroform: 573 g/L) and ether. It is sparingly soluble in aliphatic hydrocarbons (hexane: 0.1 g/L and water (pH 3: 0.0047 g/L)9. The molecule does not contain any chromophore and its UV-absorption maximum is 192 nm. Tacrolimus acts via inhibition of calcineurin. Its mechanism of action has been reviewed in references10,11. It is currently used in more than 80% of solid-organ transplant patients in the United States12.
The therapeutic index of tacrolimus is considered to be narrow13. In addition, the correlation between tacrolimus doses and blood concentrations is poor and pharmacokinetics is variable14,15. Therapeutic drug monitoring to guide tacrolimus dosing in transplant patients is therefore general clinical practice16-20. The goal is to keep the tacrolimus blood concentrations within a pre-defined therapeutic range. Tacrolimus blood concentrations below the therapeutic range may result in increased activity of chronic or acute allo-immune reactions, while concentrations above the therapeutic window increase the risk for over-immunosuppression, cancer and toxicities, such as nephrotoxicity, neurotoxicity, hypertension, and diabetes. High pharmacokinetic intra-individual variability of tacrolimus may be detrimental to both transplant organ and patient survival21,22. While inter-individual variability of tacrolimus pharmacokinetics is mainly caused by CYP3A5 polymorphisms, reasons for intra-individual variability include, but are not limited to, drug-drug, disease-drug and food-drug interactions14,15. Also lack of adherence to the immunosuppressive therapeutic drug regimen is a contributing factor and a major reason for graft loss23,24.
These considerations suggest that frequent home therapeutic drug and adherence monitoring of tacrolimus whole blood concentrations may be beneficial to ensure that patients have tacrolimus exposure within the desired therapeutic window at all times. However, the logistics and cost of more frequent therapeutic drug monitoring as it is current clinical practice15 is prohibitive. One of the reasons is that the patient has to see a phlebotomist to have the required venous blood sample drawn. Dried blood spots have recently emerged as an attractive concept25-28. After a simple finger stick the patient collects a blood drop on a special filter paper card and after the blood spot has dried, it can be mailed to a central laboratory for analysis of tacrolimus and any other immunosuppressant that the patient may currently be taking. This has become possible due to the development of highly sensitive and specific LC-MS/MS assays for the quantification of tacrolimus and other immunosuppressants in very small blood volumes such as dried blood spots (typically 20 &#181;l of blood)25,29-43. Another advantage is that minimally invasive, low volume sample collection strategies such as dried blood spots greatly facilitate therapeutic drug monitoring and pharmacokinetic studies in small children28.
Tacrolimus is usually measured in venous EDTA whole blood15. Reasons are that tacrolimus extensively distributes into blood cells and that clinical studies have reported better correlation between tacrolimus trough concentrations in blood than in plasma with clinical events15,18. In comparison, the analysis of tacrolimus in dried blood spots is based on capillary blood that is mixed with the filter paper matrix. This presents challenges in terms of solubilization of tacrolimus and potential interferences with the LC-MS/MS analysis. Here we present an established and validated assay based on homogenization of the dried blood spot using a bullet blender in combination with a high-flow online column sample clean up procedure and LC-MS/MS analysis. As of today, this assay has successfully been used for the quantification of more than five thousand tacrolimus dried blood spot samples for adherence monitoring in clinical trials.

<table><tbody><tr><td>Tacrolimus</td><td>U.S. Pharmacopeial Convention</td><td>1642802</td><td></td></tr><tr><td>D&lt;sub&gt;2&lt;/sub&gt;,&lt;sup&gt;13&lt;/sup&gt;C-Tacrolimus</td><td>Toronto Research Chemicals Inc.</td><td>F370002</td><td></td></tr><tr><td>Red blood cells</td><td>University of Colorado Hospital</td><td>W20091305500 V</td><td></td></tr><tr><td>Plasma</td><td>University of Colorado Hospital</td><td>W2017130556300Q</td><td></td></tr><tr><td>Acetone CHROMASOLV, HPLC, &amp;ge;99,9%</td><td>Sigma-Aldrich</td><td>439126-4 L</td><td></td></tr><tr><td>Acetonitrile Optima LC/MS, UHPLC-UV</td><td>Thermo Fisher Scientific</td><td>A955-4</td><td></td></tr><tr><td>Isopropanol 99.9%, HPLC</td><td>Fisher Scientific</td><td>BP2632-4</td><td></td></tr><tr><td>Methanol Optima LC/MS</td><td>Thermo Fisher Scientific</td><td>A452-4</td><td></td></tr><tr><td>Water Optima LC/MS, UHPLC-UV</td><td>Thermo Fisher Scientific</td><td>W6-4</td><td></td></tr><tr><td>Formic acid</td><td>Thermo Fisher Scientific</td><td>A118P-500</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Zinc sulfate</td><td>Thermo Fisher Scientific</td><td>Z68-500</td><td></td></tr><tr><td>0.5 &amp;ndash; 10 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008649</td><td></td></tr><tr><td>1.5 ml Eppendorf tube</td><td>Thermo Fisher Scientific</td><td>02-682-550</td><td></td></tr><tr><td>10 &amp;ndash; 100 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008651</td><td></td></tr><tr><td>10 &amp;mu;l pipet tips with filter, sterile</td><td>Neptune</td><td>BT 10XLS3</td><td></td></tr><tr><td>100 &amp;ndash; 1,000 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008653</td><td></td></tr><tr><td>100 &amp;mu;l pipet tips with filter, sterile</td><td>Neptune</td><td>BT 100</td><td></td></tr><tr><td>1,000 &amp;mu;l pipet tips with filter, sterile</td><td>Multimax</td><td>2940</td><td></td></tr><tr><td>2 &amp;ndash; 20 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008650</td><td></td></tr><tr><td>2 ml Eppendorf tube</td><td>Thermo Fisher Scientific</td><td>02-681-258</td><td></td></tr><tr><td>20 &amp;ndash; 200 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008652</td><td></td></tr><tr><td>20 &amp;mu;l pipet tips with filter, sterile</td><td>GeneMate</td><td>P-1237-20</td><td></td></tr><tr><td>200 &amp;mu;l pipet tips with filter</td><td>Multimax</td><td>2938T</td><td></td></tr><tr><td>200 &amp;mu;l pipet tips with filter, sterile</td><td>Multimax</td><td>2936J</td><td></td></tr><tr><td>50 ml Falcon tube</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>300 &amp;mu;l inserts for HPLC vials</td><td>Phenomenex</td><td>ARO-9973-13</td><td></td></tr><tr><td>Balance PR2002</td><td>Mettler Toledo</td><td>1117050723</td><td></td></tr><tr><td>Balances AX205 Delta Range</td><td>Mettler Toledo</td><td>1119343379</td><td></td></tr><tr><td>Bullet Blender Homogenizer</td><td>Next Advance</td><td>BBX24</td><td></td></tr><tr><td>Centrifuge Biofuge Fresco</td><td>Heraeus</td><td>290395</td><td></td></tr><tr><td>Disposable Wipes</td><td>PDI</td><td>Q55172</td><td></td></tr><tr><td>Glass v ials, 4 ml</td><td>Thermo Fisher Scientific</td><td>14-955-334</td><td></td></tr><tr><td>Glass vials, 20 ml</td><td>Thermo Fisher Scientific</td><td>B7800-20</td><td></td></tr><tr><td>Gloves, nitrile</td><td>Titan Brand Gloves</td><td>44-100S</td><td></td></tr><tr><td>HPLC vials, 9 mm, 2 ml, clear</td><td>Phenomenex</td><td>ARO- 9921-13</td><td></td></tr><tr><td>Lids for HPLC vials</td><td>Phenomenex</td><td>ARO- 8952-13-B</td><td></td></tr><tr><td>Needle, 18 G 1.5</td><td>Precision Glide</td><td>305196</td><td></td></tr><tr><td>Rack for Eppendorf tubes</td><td>Thermo Fisher Scientific</td><td>03-448-11</td><td></td></tr><tr><td>Rack for HPLC Vials</td><td>Thermo Fisher Scientific</td><td>05-541-29</td><td></td></tr><tr><td>Steel beads 0.9 &amp;ndash; 2 mm</td><td>Next Advance</td><td>SSB14B</td><td></td></tr><tr><td>Storage boxes for freezers / refrigerators</td><td>Thermo Fisher Scientific</td><td>03-395-464</td><td></td></tr><tr><td>Standard multi-tube vortexer</td><td>VWR Scientific Products</td><td>658816-115</td><td></td></tr><tr><td>Whatman Paper, 903 Protein Saver US 100/PK</td><td>GE Whatman&amp;nbsp;</td><td>2016-05</td><td></td></tr><tr><td>Autosampler</td><td>CTC PAL&amp;nbsp;</td><td>PAL.HTCABIx1</td><td></td></tr><tr><td>Binary pump, Agilent 1260 Infinity</td><td>Agilent Technologies</td><td>1260 G1312B</td><td></td></tr><tr><td>Binary pump, Agilent 1290 Infinity</td><td>Agilent Technologies</td><td>1290 G4220A</td><td></td></tr><tr><td>Micro vacuum degasser, Agilent 1260</td><td>Agilent Technologies</td><td>1260 G13798</td><td></td></tr><tr><td>Column oven,&amp;nbsp; Agilent 1290 with 2 position&amp;nbsp;</td><td>Agilent Technologies</td><td>1290 G1216C</td><td></td></tr><tr><td>Thermostated column compartment with integrated 6 port switching valve</td><td>Agilent Technologies</td><td>1290 G1316C</td><td></td></tr><tr><td>HPLC pre-column cartridge, Zorbax XDB C8 (5 &amp;micro;m particle size), 4.6 &amp;middot; 12.5 mm</td><td>Phenomenex</td><td>820950-926</td><td></td></tr><tr><td>HPLC analytical column, Zorbax Eclipse-XDB-C8 (5 &amp;micro;m particle size), 4.6 &amp;middot; 150 mm</td><td>Phenomenex</td><td>993967-906</td><td></td></tr><tr><td>Tandem Mass Spectrometer</td><td></td><td></td><td></td></tr><tr><td>API5000 MS/MS with TurboIonspray source</td><td>AB Sciex</td><td>4364257</td><td></td></tr><tr><td>Mass spectrometry software</td><td>AB Sciex</td><td>Analyst 1.5.1</td><td></td></tr></tbody></table>

quantification of the immunosuppressant tacrolimus on dried blood spots using lc-ms/ms

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Watch this Scientific Journal Video about Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS at JoVE.com

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

quantification-immunosuppressant-tacrolimus-on-dried-blood-spots

Nanyang Technological University

Research

JoVE Journal

Medicine

16.8K Views.  University of Colorado, Anschutz Medical Campus. Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

Video: Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

Cancer Research

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Bioengineering

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Immunology and Infection

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Chemistry

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Biochemistry

Evaluation of Drug Sorption to PVC- and Non-PVC-based Tubes in Administration Sets Using a Pump

Administration sets are delivery tools for the direct application of drugs into the body and are composed of a spike, a drip chamber, tubes, Luer adapters (connectors), a needle cover for protection, and other accessories. Drug sorption to tubes of administration sets is a critical issue in terms of safety and efficacy. Although drug sorption is an important factor in the quality of an administration set, there are no standard evaluation methods for the regulation of drug sorption to the tubes. Here, we describe an evaluation protocol for drug sorption to tubes of administration sets. Tubes made of polyvinyl chloride (PVC)- and non-PVC-based polymeric materials were cut to 1 m in length. Diazepam and tacrolimus were used as model drugs. In the kinetic sorption study, we selected the drug concentration and flow rate based on the clinical usage of these drugs. After the dilution of each drug in a glass bottle, the diluted drug solution was delivered through tubes of administration sets using a pump. Samples were collected in amber vials at appropriate time points and the drugs were analyzed using high-performance liquid chromatography. Drug concentrations and sorption levels to tubes of the administration sets were calculated. Acceptable criteria to ensure the quality of administration sets are recommended.

We report a method for drug sorption evaluation using a pump and model drugs (diazepam and tacrolimus). After drug analysis using high-performance liquid chromatography, drug concentrations and sorption levels in the tubes of administration sets are calculated.

Administration sets are delivery tools for the direct application of drugs into the body and are composed of a spike, a drip chamber, tubes, Luer adapters ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Biology

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

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